Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Reexamination Certificate
1999-06-30
2003-09-09
Nolan, Patrick J. (Department: 1644)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
C530S387300, C530S389700, C530S388800, C424S184100, C435S328000, C435S330000, C435S331000, C435S810000
Reexamination Certificate
active
06617434
ABSTRACT:
FIELD OF THE INVENTION
The present invention provides Methyl- (or Mutant-) Differential Display (MDD) methods and nucleic acid probes for detecting mutations and/or the methylation patterns of nucleic acids. Genes are frequently not methylated in the cells where they are expressed but are methylated in cell types where they are not expressed. Moreover, tumor cell DNA is frequently methylated to a different extent and in different regions than is the DNA of normal cells. The present invention is used for identifying which regions of the genome are methylated or mutated in different cell types, including cancerous cell types. The present invention also is used for identifying whether a tissue sample has a methylation or mutation pattern which is normal or like that of known cancer cells. The present invention can be used to identify aberrant expression of known or unknown genes. Such aberrant expression may occur at an incorrect location or an incorrect time, i.e. tissue or developmental specificity is lost. Genes and genomic regions discovered according to the present invention are useful for identifying products, e.g. mRNA, protein or fragments thereof, which are aberrantly expressed.
BACKGROUND OF THE INVENTION
DNA is often methylated in normal mammalian cells. For example, DNA is methylated to determine whether a given gene will be expressed and whether the maternal or the paternal allele of that gene will be expressed. (Little and Wainwright, 1995). While methylation is known to occur at CpG sequences, only recent studies indicate that CpNpG sequences may be methylated (Clark et al., 1995). Methylation at CpG sites has been much more widely studied and is better understood.
Methylation occurs by enzymatic recognition of CpG and CpNpG sequences followed by placement of a methyl (CH
3
) group on the fifth carbon atom of a cytosine base. The enzyme that mediates methylation of CpG dinucleotides, 5-cytosine methyltransferase, is essential for embryonic development—without it embryos die soon after gastrulation. It is not yet clear whether this enzyme methylates CpNpG sites (Laird and Jaenisch, 1994).
When a gene has many methylated cytosines it is less likely to be expressed (Willson, 1991). Hence, if a maternally-inherited gene is more highly methylated than the paternally-inherited gene, the paternally-inherited gene will generally give rise to more gene product. Similarly, when a gene is expressed in a tissue-specific manner, that gene will often be unmethylated in the tissues where it is active, but will be highly methylated in the tissues where it is inactive. Incorrect methylation is thought to be the cause of some diseases including Beckwith-Wiedemann syndrome and Prader-Willi syndrome (Henry et al., 1991; Nicholls et al., 1989).
The methylation patterns of DNA from tumor cells are generally different than those of normal cells (Laird, 1994). Tumor cell DNA is generally undermethylated relative to normal cell DNA, but selected regions of the tumor cell genome may be more highly methylated than the same regions of a normal cell's genome. Hence, detection of altered methylation patterns in the DNA of a tissue sample is an indication that the tissue is cancerous. For example, the gene for Insulin-Like Growth Factor 2 (IGF2) is hypomethylated in a number of cancerous tissues, such as Wilm's Tumors, rhabdomyosarcoma, lung cancer and hepatoblastomas (Rainier et al., 1993; Ogawa et al., 1993; Zhan et al., 1994; Pedone et al., 1994; Suzuki et al., 1994; Rainier et al., 1995).
The present invention is directed to a method of detecting differential methylation at CpNpG sequences by cutting test and control DNAs with a restriction enzyme that will not cut methylated DNA, and then detecting the difference in size of the resulting restriction fragments.
While methylation-sensitive restriction enzymes have been used for observing differential methylation in various cells, no commercial assays exist for use on human samples because differentially methylated sequences represent such a minute proportion of the human genome that they are not readily detected. The human genome is both highly complex, in that it contains a great diversity of DNA sequences, and highly repetitive, in that it contains a lot of DNA with very similar or identical sequences. The high complexity and repetitiveness of human DNA confounds efforts at detecting and isolating the minute amount of differentially methylated DNA which may be present in a test sample. The present invention remedies this detection problem by providing new procedures for screening a selected subset of the mammalian genome which is most likely to contain genetic functions.
The present invention provides techniques for detecting and isolating differentially methylated or mutated segments of DNA which may be present in a tissue sample in only minute amounts by using one or more rounds of DNA amplification coupled with subtractive hybridization to identify such segments of DNA. DNA amplification has been coupled with subtractive hybridization in the Representational Difference Analysis (RDA) procedures disclosed in U.S. Pat. No. 5,436,142 to Wigler et al. and Lisitsyn et al. (1993). However, for the subtractive hybridization step of such RDA procedures to proceed in a reasonable time and with reasonable efficiency, only a subset of the genome can be examined. To accomplish this necessary reduction in the complexity of the sample DNA, Wigler et al. and Lisitsyn et al. disclose cutting DNA samples with restriction enzymes that cut infrequently and randomly. However, selection of enzymes which randomly cut the genome means that the portion of the genome which is examined is not enriched for any particular population of DNA fragments. Thus, when RDA is used, only a random subset of the human genome, which includes repetitive elements, noncoding regions and other sequences which are generally not of interest, can be tested in a single experiment.
In contrast, the present invention is directed to methods which use enzymes that cut frequently and that specifically cut CG-rich regions of the genome. These enzymes are chosen because CG-rich regions of the genome are not evenly distributed in the genome—instead, CG-rich regions are frequently found near genes, and particularly near the promoter regions of genes. This means that the proportion of the genome that is examined by the present methods will be enriched for genetically-encoded sequences as well as for regulatory sequences. Moreover, unlike the RDA method, the present methods selectively identify regions of the genome which are hypomethylated or hypermethylated by using enzymes which specifically cut non-methylated CG-rich sequences. The present invention therefore represents an improvement over RDA methods because of its ability to select DNA fragments which are likely to be near or to encode genetic functions.
SUMMARY OF THE INVENTION
The present invention provides probes and methods of detecting whether a CNG triplet is hypomethylated or hypermethylated in a genomic DNA present in a test sample of cell.
The present invention provides a method of detecting whether a CNG triplet is hypomethylated or hypermethylated in a genomic DNA present in a test sample of cells which includes:
a) isolating genomic DNA from a control sample of cells and a test sample of cells to generate a control-cell DNA and a test-cell DNA;
b) cleaving the control-cell DNA and the test-cell DNA with a master restriction enzyme to generate cleaved control-cell DNA and cleaved test-cell DNA;
c) preparing a probe from a DNA isolated by the present methods, for example, a DNA selected from the group consisting of SEQ ID NO:7-10;
d) hybridizing the probe to the cleaved control-cell DNA and the cleaved test-cell DNA to form a control-hybridization complex and a test-hybridization complex; and
e) observing whether the size of the control-hybridization complex is the same as the size of the test-hybridization complex;
wherein the master restriction enzyme cleaves a nonmethylated CNG DNA sequence but does not cleave a met
Kenyon & Kenyon
Nolan Patrick J.
North Shore - Long Island Jewish Research Institute
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